Role Of Pea3 In The Development Of Kidney And Its Possible Mechanism

Renal disease is one of the most common diseases in children. It has been demonstrated that the repair process of kidney after damage is similar to the developing bioresearch. So investigating the nephrogenesis from the developmental biology will be benifitial for studying the mechanism of renal dysplasia and disease, and recently the development of kidney attract more and more attention.We start to study the development of kidney from 2003, applied the suppression subtractive hybridization to build the neonatal renal and post-natal day 21 renal subtractive library to screen the new developmental genes. We found that the expression of pea3 (polyomavirus enhancer activator3) was upregulated in the neonatal renal subtractive library by bioinformatics.Transcription factor pea3 belongs to the PEA3 subgroup of the Ets family, and Ets family proteins have been shown to play an important role in different biosystems , including regulating the growth, transcription, the activation of T cell, and the development of organs and so on. The expression of pea3 mainly located in the murine developing organs, especially in epithelial–mesenchymal interaction events, for example, kidney, lung and beast, which indicates that it plays a key role in these organogenesis. However, the role of pea3 in kidney development has not yet been clarified. Recent study has showed the close relationship between pea3 and other two moleculars (wnt and wt1, both of them are important to the kidney development). Here we firstly observe the spatial and temporal expression of pea3 during the development of rat kidney, then study it's role and the relationship among pea3, wnt and wt1 by stimulation with wnt3a, overexpression of pea3 expression vector, and downregulation of pea3 by siRNA, respectively. We also apply zebrafish as the model to study the action and mechanism of pea3 in kidney development in vivo by micro-injection of pea3 mRNA or downregulation of pea3. Part I: The temporal and spatial expression pattern of pea3 in rat kidney developmentObject To explore the temporal and spatial expression pattern of pea3 in rat kidney development.Methods Kidneys were dissected from embryos at E13, E15, E17 and E19, and from postnatal days P0, P7, P14, P21 and adult rats. Expression of pea3 was evaluated by real-time RT-PCR (polymerase chain reaction), western blot and in situ hybridization analysis.Results Both real-time RT- PCR and western blot analyses revealed that pea3 exhibited dynamic developmental regulation, with high levels of expression from embryonic day E15 until birth, and declining levels thereafter. By in situ hybridization, pea3 mRNA was detected in the ureteric bud (UB) and surrounding metanephric mesenchyme of the kidneys from E15 until birth, but was undetectable in mature kidneys.Conclusion These studies suggest that the newly identified gene pea3 may be involved in rat kidney development and differentiation, specially during the epthelial-mesenchymal interaction.Part II: Role and mechanisms of pea3 played in rat kidney developmentObject To develop the role of pea3 played in rat kidney development and it's mechanisms.Methods (1) The expression of pea3 in the different tissues of neonatal rat was investigated by real-time RT-PCR; (2) Pea3 and wt1 expression patterns during the rat kidney development was detected by double-immunofluorescence analysis; (3) RIMM-18 cells were incubated with wnt3a (100ng/ml) for 0-2 days, then the change of morphology was detected and the expression of pea3, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively; (4) The pea3 expression vector was cloned, then transfected into RIMM-18 cells, the change of morphology was detected and the expression of pea3, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively. Cells treated with wnt3a were used as the positive control, then observe cells'morphology and detect the expression of wt1, E-cadherin and vimentin by western blot; (5) The pea3 siRNA was synthesized, then transfected into RIMM-18 cells in the presence or absence of wnt3a, and the change of morphology was detected and the expression of pea3,β-catenin, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively; (6) To develop the signal pathway of wnt3a regulating pea3, RIMM-18 cells were pretreated with U0126 or wnt/β-catenin specific inhibitor DKK-1, then wnt3a was added, the expression ofβ-catenin, p-ERK, pea3 and wt1 were analysized by immunoblotting for investigating the signaling pathway involved in wnt3a-regulated pea3.Results (1) The expression of pea3 in various tissues of newborn rat by SYBR green real-time RT-PCR analysis showed that pea3 is highest in kidney, lower in lung, pancreas and brain, little in spleen and liver; (2) Double-immunofluorescence staining for pea3 and wt1 showed the expression of pea3 was similar to the result of ISH before, and the expression of wt1 has many overlaps with that of pea3. The concordant expression of pea3 and wt1 in the condensing metanephric mesenchyme and the ureteric branches was showed; (3) Real-time RT-PCR data indicated that, incubation with wnt3a for 2 days led to an increase of pea3 mRNA at 6h and a decrease tendency in vimentin mRNA. By western blot, both pea3 and wt1 were obviously upregulated after stimulated with wnt3a for 12 hours, and E-cadherin was significantly increased after 2 days treatment. The morphologic changes were assessed by phase contrast microscopy. RIMM-18 cells exhibited cobble-stone-like epithelial morphology after 2 days incubation with wnt3a; (4) pea3 had increased wt1 and E-cadherin, and downregulated vimentin expression in western blot analysis after the cells transfected with pea3 expression vector, and the cells showed cobble-stone-like epithelial morphology change, which were also similar to the results of treated with wnt3a; (5) Firstly, real-time PCR showed Pea3 siRNA no. 885 could decreased pea3 expression at 24h post-transfection, then western blot analyses revealed that wnt3a stimulation treatment increased pea3, wt1, and E-cadherin and reduced vimentin expression respectively, and these expressions were reduced in wnt3a-treated cells transfected with pea3 siRNA; (6) Western blot analyses revealed that ERK1/2 was activated after treated by incubation with wnt3a for 3h and 6h, when pretreated with U0126, the ERK1/2 specific inhibitor, blocked the increase of both pea3 and wt1 induced by wnt3a.β-catenin was also increased after treated with wnt3a. While pretreated pretreatment with Dkk-1 prior to the addition of wnt3a,β-catenin was reduced, neither p-ERK, pea3 nor wt1 expression had no obviously changed.Conclusion The data identify that: (1) wt1 is critical for kidney development, and pea3 may be as a wt1 trans-acting factor, important for mesenchymal-epithelial transitions. (2) wnt3a can induces pea3 through activation of ERK1/2 signaling pathway, however, there are maybe another cross-talk pathways during the proceed.Part III: Expression and function of the Ets transcription factor pea3 during the formation of the zebrafish pronephrosObject To observe the temporal and spatial expression pattern of pea3 during formation of the zebrafish pronephros, and explore the roles of pea3 in pronephrogenesis, which further verify the results before, and provide clues to pathogensis of congenital renal disease and targets for therapy.Methods (1) zebrafish embryos at different stages (25hpf, 36hpf, 51hpf) (hpf: hour post fertilization) were collected for whole mount double in situ hybridization and mesonephros were isolated from muture zebrafish for RT-PCR; (2) zebrafish embryos were microinjected pea3 mRNA for overexpression of pea3, then the markers of zebrafish pronephros - wt1a, podocin, pax2.1 and cdh17 were detected at 24hpf and 48hpf by in situ hybridization; (3) pea3/erm morpholinos were applied to knockdown the expression of pea3 and erm, respectively, then the expression of zebrafish pronephros markers were examined at 24hpf and 48hpf by in situ hybridization. The expression of wt1a and podocin was also detected at 7dpf after microinjection of pea3/erm morpholinos. Furthermore, pea3 mRNA was microinjected in addition with pea3/erm morpholinos, then the expression of zebrafish pronephros marker genes were assessed at 24hpf and 48hpf by in situ hybridization; (4) wt1a mRNA was microinjected in addition with pea3/erm morpholinos, then the expression of podocin and cdh17 was detected at 48hpf by in situ hybridization.Results (1) By double in situ hybridization, pea3 mRNA was undetectedable at 25 hpf in pronephron, while wt1a is abundant in pronephron primordia. Both pea3 and wt1a expressed in glomerular primordia at 36 hpf, the area of pea3 expression was smaller than wt1a. Pea3 was expressed in pronephric glomerular at 51 hpf and had overlappping expression with wt1a. Pea3 also expressed in mature zebrafish mesonephros by RT-PCR; (2) Overexpression of pea3 by pea3 mRNA, embryos delayed the development. The distribution of wt1a and podocin was abnormal by in situ hybridization. Glomerular primordia of both sides were abnormal fusion. In addition, AP staining showed the distance between the two anteriors of pronephric tubulars also became larger, while the expression of pax2.1 and cdh17 had no obviously change; (3) Simultaneous microinjection of MO-pea3 and MO-erm could lead to the delayed development of embryos, some embryos appared pericardial edema, and in situ hybridization analysis showed the area of wt1a and podocin expression was reduced notablely. Moreover, the width between the two glomerulars was enlarged, and could not fuse normally at 48 hpf. However, the expression of cdh17 and pax2.1 was not reduced strikingly. While injected with single MO-pea3 or MO-erm, the development of embyos had no signally affected. In addition, when simultaneous microinjected of pea3/erm co-morpholinos with pea3 mRNA, the defects caused by pea3/erm co-morpholino were rescued; (4) Microinjection of wt1a mRNA in addition with pea3/erm co-morpholinos, the expression of podocin almost returned to the normal by in situ hybridization.Conclusion In summary, we firstly observed pea3 expressed in zebrafish pronephron, and the pea3 subgroup of Ets family, including pea3 and erm, were required for the pronephrogenesis maybe through wt1a.